The dominantly inherited thyroid cancer syndromes multiple endocrine neoplasia types 2A, 2B (MEN-2A, MEN-2B) and familial medullary thyroid carcinoma (FMTC) have been mapped by genetic linkage analysis to the pericentromeric region of human chromosome 10. The ultimate goal of the proposed research is to clone the gene or genes responsible for the disorders and determine the molecular basis of the mutations that give rise to these complex phenotypes. We plan to clone the genomic DNA that comprises the interval between genetic markers flanking the MEN-2 locus on chromosome 10 by a chromosome walking approach. Our primary resources for the walk will be a human genomic yeast artificial chromosome (YAC) library and a bacteriophage P1 genomic library constructed from a somatic cell hybrid containing human chromosome 10. Sequential bidirectional walking from the ends of YACs identified with probes to closely linked loci will be augmented by a more general approach of STS (sequence tagged site) content mapping using a large number of clones that map to the interval. Limited physical mapping studies will be done to construct clone contigs and to identify regions of interest to be tested for the presence of genes. New genetic markers will be developed and tested for linkage to more precisely define the placement of the disease loci. These new markers will also serve as reagents for the development of a presymptomatic linkage-based diagnostic test which will be more that 99% accurate and informative for nearly all individuals at risk for the disorders. We plan to make use of cDNA libraries available from several sources in order to identify candidate genes that map to the pericentromeric region of chromosome 10. Gene candidates will be characterized by a variety of approaches including testing for the presence of chromosomal rearrangements or deletions using a panel of DNAs from MEN-2 affected individuals. Once the gene(s) are cloned and mutations fully characterized we will continue efforts to develop direct DNA diagnostic tests for these disorders.